Non-viral gene delivery systems must overcome the following sequential "barriers" in order to effectively deliver genetic material. 1) Effective self-assembly of the delivery system with the genetic material, 2) stability of the delivery system in the extracellular (serum and blood) environment, 3) escape from the endosomal/lysosomal compartments of the cell, and 4) delivery into the nucleus without the requirement for cell division. We have found that no single barrier is predominant. Instead, effective non-viral gene delivery systems will become therapeutic realities only with a multidisciplinary research effort that addresses all four of these barriers in an integrated fashion, and only with an integrated delivery system with chemical and biophysical properties that allow all these barriers to be overcome. In this regard, we have developed a delivery system that has several extraordinary features, making it a highly attractive candidate for systemic administration in mammals. These features include resistance to inactivation by serum, releasable serum protection components, multiple endosomal escape mechanisms with low cytotoxicity, and most uniquely, the ability to transfect cells equally well with or without cell division. For this project, we have assembled a multidisciplinary team to address the sequential barriers to non-viral gene delivery in an integrated fashion. 1) Effective self-assembly will be achieved by developing fully automated microfluidic technology to carry out reconstitution, mixing, dialysis, and concentration. Stability in serum will be achieved by polyethyleneglycol (PEG) stabilization, with triggered release of the PEG chains upon entry into endosomal compartments. Endosomal escape will be achieved with multiple membrane fusion mechanisms, including fusogenic peptide mimetics designed to eliminate protease inactivation and immune response. Nuclear entry, without cell division, will be achieved with nuclear targeting signals that by-pass classical nuclear targeting pathways, and instead bind directly to the nuclear pore - a mechanism used by those viruses that are the most effective at nuclear entry without cell division. These signals will also be in the form of peptide mimetics to eliminate protease degradation and immune response. The development of this technology will have broad implications for the controlled self-assembly of a wide range of nanoparticle systems, particularly those intended for in vivo drug delivery. [unreadable] [unreadable]